TGFβ Family
TGFβ1 is the prototypical member of a family of cytokines important in the regulation of phenomena including cell growth, differentiation, apoptosis and morphogenesis in species from insects to mammals. Members of the TGFβ family, include the TGFβs, Activin, Bone Morphogenetic Proteins (BMP's) and Mullerian Inhibitory Substance. These cytokines share structural characteristics, usually occur as disulfide-linked homodimers, and signal via structurally similar plasma membrane receptors (reviewed in Massagué et al., 1997, Trends Cell Biol., 7: 187-192). TGFβ family receptors are distinct from most known plasma membrane receptor kinases in that they are serine/threonine kinases. Based on sequence similarities, the TGFβ family receptors fall into two categories, termed type I and type II, are functionally different. Type I receptors are distinguished from type II receptors by inclusion of a conserved region termed a GS domain, located in the juxtamembrane intracellular region, which is rich in glycine and serine residues. Both type I and type II receptors are necessary for signaling (reviewed in Heldin, 1997, Nature,390: 465-471). Each receptor type exists in the plasma membrane in an oligomeric form (Henis et al., 1994, J. Cell Biol., 126: 139-154; Chen & Derynck, 1994, J. Biol. Chem. 269: 22868-22874).
Ligand binding to the type II receptor activates the receptor kinase activity (Wrana et al., 1994, Nature, 370: 341-347). The activated receptor-ligand complex then recruits the type I receptor, activating it by phosphorylation of the GS domain (Wrana et al., 1994, supra; Weiser et al., 1995, EMBO J. 14: 2199-2208). The ligand-bound, active receptor complex is thought to be a heterotetramer, containing taco type II and two type I receptor molecules (Yamashita et al., 1994, J. Biol. Chem., 269: 20172-20178). Overexpression and mutagenesis studies have indicated that the type I receptor acts downstream of the type II receptor. Thus, the identity of the type I receptor determines the specificity of the signals generated (Carcamo et al., 1994, Mol. Cell. Biol., 14: 3810-3821).
Smads
Until recently, little was known of the signaling mechanisms and intracellular effector proteins mediating the complex responses following TGFβ family receptor activation. This changed with the discovery of Mad, a Drosphila protein which suppressed a mutation in the BMP2/BMP4 homolog Decapentaplegic (Sekelsky et al., 1995, Genetics, 139: 1347-1358). Mad was found to act downstream of the Drosophila type I receptor, Thick veins (Wiersdorff et al., 1996, Development, 122: 2153-2162). Similarly, C. Elegans proteins termed Sma2, Sma3 and Sma4 were found to act downstream of, and to be required for, signaling through the BMP2/BMP4 homolog Daf4. Sma2, Sma3 and Sma4 are structurally related to each other and to Mad (Savage et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 790-794). A family of at least nine factors, termed Smads, have been identified from Xenopus to man and shown to be components of the signaling pathway downstream of TGFβ receptors.
Smads are of a relative molecular weight of 42 to 60 kD, and bear two regions of homology, termed MH1 and MH2, located at the amino and carboxy-terminal ends of the molecule, respectively. A proline-rich linker separates MH1 and MH2 (Heldin et al., 1997, supra). Different members of the Smad family have different roles in signaling. Smad1, Smad2, Smad3, Smad5 and possibly Smad8 are activated by specific type I receptors. They are thus referred to as ligand-responsive, or pathway restricted Smads. For example, Smad1, Smad5 and possibly Smad9 function downstream of BMP type I receptors, while Smad2 and Smad3 are activated by TGFβ and Activin type I receptors (Hoodless et al., 1996, Cell, 85: 489-500; Graf et al, 1996, Cell, 85: 479-487; Thomsen et al, 1996, Development, 122: 2359-2366; Yamamoto et al., 1997, Biochem. Biophys. Res. Commun., 238: 574-580; Baker & Harland, 1996, Genes Dev., 10: 1880-1889; Eppert et al., 1996, Cell, 86: 543-552; Lechleider et al., 1996, J. Biol. Chem., 271: 17617-17620; Yingling et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 8490-8944; Zhang et al., 1996, Nature, 383: 168-172; Nakao et al., 1997, EMBO J., 16: 5353-5362). In contrast to the pathway restricted Smads, Smad4, also known as DPC4, functions as a common signaling partner, interacting with all ligand-responsive Smads. Following activation by type I receptors, pathway restricted Smads, cytoplasmically located in their non-activated state, rapidly translocate to the nucleus. This nuclear localization is mediated by association of pathway restricted Smads with the common mediator Smad4 (Zhang et al., 1996, supra; Kretzschmar et al., 1997, Genes Dev., 11: 984-995; Lagna, et al., 1996, Nature, 383: 832-836; Wu et al., 1997, Mol. Cell. Biol., 17: 2521-2528).
The actual mechanism of activation of pathway restricted Smads is thought to involve direct phosphorylation of the three most C-terminal serine residues in a Ser-Ser-X-Ser motif, located near the C terminus of the MH2 region, by activated type I receptors (Kretzschmar et al., 1997, supra; Marcias-Silva et al., 1996, Cell, 87: 1215-1224; Souchelnytskyi et al., 1997, J. Biol Chem., 272: 28107-28115; Abdollah et al., 1997, J. Biol. Chem., 272: 27678-27685). Following phosphorylation of MH2, the pathway restricted Smads form heteromeric complexes with Smad4.
There is evidence that MH1 and MH2 interact when Smads are in the inactive state. It is thought that MH2 phosphorylation by type I receptors may destabilize the intramolecular interaction of MH1 and MH2, thereby allowing Smad 4 to interact with the MH2 domains of ligand responsive Smads (Souchelnytskyi et al., 1997, supra; Hata et al., 1997, Nature, 388: 82-87). This implies that MH1 is an inhibitory domain, an implication supported by the identification of MH1 mutations in Smad2 and Smad4 in certain tumors which result in strengthened affinity of the MH1 domain for the corresponding MH2 domain (Eppert et al., 1996, supra; Schutte, 1996, Cancer Res., 56: 2527-2530; Hata et al., 1997, supra).
In addition to its likely role in regulating Smad4 interaction, the MH2 region of pathway restricted Smads may serve as an effector domain in signal transduction. This is suggested by the ability of MH2 to transactivate a transcriptional response when fused to the yeast Gal4 DNA binding domain (Liu, 1996, Nature, 381: 620-623), and by the finding that an isolated MH2 domain derived from Smad2 supports a full range of Activin responses (Baker & Harland, 1996, supra). In Smad4, however, a portion of the linker between MH1 and MH2, not conserved to other Smads, is required for signaling in addition to MH2 (deCaestecker et al., 1997, J. Biol. Chem., 272: 13690-13696).
The nuclear translocation of the pathway-restricted Smad:Smad4 complexes, and the ability of some Smads to associate with DNA-binding factors or to directly bind to specific DNA sequences suggest Smads may have transcriptional regulatory functions (Chen et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 12992-12997; Chen et al., 1997, Nature, 389: 85-89; Liu et al., 1996, supra; Kim et al., 1997, Nature, 388: 304-308; Yingling et al., 1997, Mol. Cell. Biol., 17: 7019-7028; Dennler et al., 1998, EMBO J., 17: 3091-3100; Zawel et al., 1998, Mol. Cell, 1: 611-617). However, it is not clear how Smads regulate transcription or whether Smads also have other nuclear functions.
Proteasome-mediated Protein Degradation Pathway
It has been demonstrated that key events within the cell are regulated by controlled degradation of proteins. In particular, it has been demonstrated that the ubiquitin (Ub)-proteasome pathway plays a critical role in the regulation of processes including the cell cycle, cell metabolism, apoptosis, signal transduction, the immune response, and protein quality control (Reviewed in Coux et al., 1996, Ann. Rev. Biochem., 65: 810-847). In the (Ub)-proteasome pathway, proteins modified by the enzymatic addition of polyubiquitin chains are rapidly and selectively degraded by large proteolytic complexes, termed proteasomes, present in both the nucleus and cytoplasm of all eukaryotic cells.
The primary component of the proteasome protein degradation pathway is the multi-subunit 20S proteasome complex, which has a molecular weight of 700-750 kD and comprises the functional proteolytic activities. Two additional multi-subunit complexes, termed the 19S (or 22S or PA700) and 11S complexes, serve regulatory functions. The overall structure of the 20S core complex is conserved from eubacteria through mammals and consists of a cylinder made up of four stacked rings (Kleinschmidt et al., 1983, Eur. J. Cell Biol., 32: 143-156; Baumeister et al., 1988, FEBS Lett., 241: 239-245). The rings are made up of protein subunits of two classes, termed α and β, each of which has seven sub-classes. The top and bottom rings of the stack are composed of α subunits, and the middle two rings are composed of β subunits. Each ring of the α or β class is itself made up of seven non-identical protein subunits, one from each respective sub-class. A number of subunits representing all sub-classes have been isolated and cloned (Coux et al., 1996, supra). All α subunits are distinguished by a conserved N-terminal motif which is necessary for ring assembly and absent from members of the β class. The α subunits assemble into rings independently, and allow the subsequent assembly of β subunit rings (Zwickl et al., 1994, Nat. Struct. Biol., 1: 765-770). Functionally, the α subunits, being located at the ends of the open cylinder, form a physical barrier preventing access of cytosolic proteins to the proteolytic core of the cylinder. In addition, the α subunits are the sites of binding by the 19S and 11S regulatory complexes. The β subunit rings make up the active proteolytic core of the 20S complex and are characterized by an N-terminal prosequence which is cleaved during assembly to reveal a threonine residue required for catalysis by the active site. The assembled 20S complex has multiple peptidase activities, but requires unfolding of protein substrates and lacks specificity for ubiquitin-modified proteins (Wilk & Orlowski, 1983, J. Neurochem., 40: 842-849; Orlowski, 1990, Biochemistry, 29: 10289-10297). Importantly, the identity of the various members of the α and β subunit rings of the 20S core may vary within a given species, among different tissues, and at different stages of development. These differences may have significant physiological impacts (Brown, 1993, J. Immunol., 151: 1193-1204; Akayama, 1994, FEBS Lett., 343: 85-88; Haass, 1989, Exp. Cell Res., 180: 243-252; Ahn, 1991, J. Biol. Chem., 266: 15746-15749; Hong, 1994, Biochem. Mol. Biol. Int., 32: 723-729; Van Kaer, 1994, Immunity, 1: 533-541).
Selectivity of the 20S proteasome for ubiquitinated proteins is conferred by the ATP-dependent association of the 20S proteasome with the 19S regulatory complex to form the 26S proteasome, which has a molecular weight of approximately 2000 kD (Yoshimura et al., 1993, J. Struct. Biol., 111: 200-211; Hoffman et al., 1992, J. Biol. Chem., 267: 22362-22368). The 19S complex contains at least 18 subunit proteins, ranging in size from 24 to 105 kD. Together, they confer a means of recognizing ubiquitinated proteins, removing the polyubiquitin chains, increasing the activity of the 20S core, and unfolding and introducing the substrate proteins to the 20S core for degradation. A number of 19S subunits have been isolated and cloned, including at least six that have ATPase activity. The reasons for the existence of so many different ATPase subunits or the specific functions of these and the other, non-ATPase subunits are not well understood (Coux et al., 1996, supra; Tanaka, 1998, Biochem. Biophys. Res. Commun., 247: 537-541).
While most substrates for the proteasome degradation pathway are modified or “tagged” by the addition of ubiquitin chains, this is not necessarily so in all cases. An alternative means of targeting proteins to the 26S proteasome pathway involves the protein antizyme, originally characterized as a naturally-occurring 26.5 kD inhibitor of ornithine decarboxylase (ODC) enzyme activity (Heller et al., 1976, Proc. Natl. Acad. Sci. USA, 73: 1858-1862). Antizyme was subsequently shown not only to inhibit ODC activity, but also to target the protein to the 26S proteasome for degradation, independently of ubiquitin (Murakami & Hayashi, 1985, Biochem. J., 226: 893-896; Hayashi & Murakami, 1995, Biochem. J., 306: 1-10). Analogous to the ubiquitin-mediated pathway, antizyme itself is removed and recycled to mediate further targeted degradation (Murakami et al., 1992, Biochem. J., 283: 661-664). Much less is known about the antizyme-dependent versus the ubiquitin-dependent degradation pathway. In particular, it is not known how many proteins other than ODC are degraded in an antizyme-dependent manner. There is a need in the art for fully understanding the signaling mechanism of Smad proteins.
There is also a need in the art for identifying proteins that are binding partners for Smad proteins.
There is also a need in the art for identifying proteasome substrates and understanding how these substrates are targeted to the proteasome.
There is also a need in the art for identifying antizyme substrates.
There is also a need in the art for identifying proteins that are binding partners for proteasome components, including ubiquitin and antizyme.
There is also a need in the art for understanding how ubiquitin and antizyme target proteins for proteasome-mediated degradation.